Decellularized bone marrow extracellular matrix

ABSTRACT

The invention is directed to compositions comprising decellularized bone marrow extracellular matrix and uses thereof. Methods for repairing or regenerating defective, diseased, damaged or ischemic tissues or organs in a subject, preferably a human, using the decellularized bone marrow extracellular matrix of the invention are also provided. The invention is further directed to a medical device, preferably a stent or an artificial heart, and biocompatible materials, preferably a tissue regeneration scaffold, comprising decellularized bone marrow extracellular matrix for implantation into a subject.

1. FIELD OF THE INVENTION

The invention relates generally to decellularized bone marrowextracellular matrix, as well as methods for the production and usethereof. In particular, the invention relates to treating defective,diseased, damaged or ischemic tissues or organs in a subject, preferablya human, by injecting or implanting decellularized bone marrowextracellular matrix into the subject. More particularly, the inventionis directed to a medical device, preferably a stent or an artificialheart, comprising decellularized bone marrow extracellular matrix forimplantation into a subject. Methods for manufacturing and coating asurface of the medical device with decellularized bone marrowextracellular matrix are also provided. The invention further relates toa biocompatible material, preferably a tissue regeneration scaffold,comprising decellularized bone marrow extracellular matrix.

2. BACKGROUND OF THE INVENTION

The healing of damaged or diseased tissues or organs is, in general,imperfect, resulting in scarring and loss of function. Theineffectiveness of pharmacological therapy has led physicians to resortto reconstructive surgery that repairs or reconstitutes soft tissue orskin by introducing an injectable or implantable material. For example,silicon gel, polyester fiber, and polytetrafluorethylene (PTFE) havebeen extensively used as implants designed to replace diseased ordamaged body parts. Other products that have been injected into thehuman body to correct soft tissue and skin defects include paraffin,petrolatum, vegetable oils, lanolin, and bees wax. Implantation orinjection of these synthetic materials, however, can become nazardous tothe health of the patient due to leakage, calcium deposit, hematoma,nodules, cellulitis, skin ulcers, and the triggering of autoimmunediseases (e.g., joint swelling and flu-like symptoms).

To reduce immune response caused by the injection or implantation ofnon-biocompatible materials, hyaluronic acid gel, a non-animalbiomaterial, has been used for soft tissue augmentation (Lupton J. andAlster T., 2000, Dermatol. Surg. 26:135-7). Bovine collagen has alsogained widespread use as an injectable material for soft tissueaugmentation. Collagen is the principal extracellular matrix structuralprotein. Early collagen implants are often solid collagen masses whichwere cross-linked with chemical agents, radiation or other means toimprove mechanical properties, decrease immunogenicity and/or increaseresistance to resorption (see, e.g., Oliver R. et al., 1976, Clin.Orthop. 115:291-30; 1980, Br. J. Exp. Path. 61:544-549; 1981, Conn.Tissue Res. 9:59-62). The main problems associated with solid implants,however, are that they must be implanted surgically and often loseflexibility due to continuing cross-linking in situ. In contrast, whileinjectable collagen implant materials might have improved volumeconsistency and resistance to physical deformation, they are expensiveand time consuming to prepare.

Also, engineered tissue has been used to augment existing tissue. Tissueengineering is an emerging field that studies the repair or regenerationof damaged or diseased tissues or failing or aging body parts withlaboratory-grown parts such as bone, cartilage, blood vessels, and skin.Tissue engineering is based upon a relatively simple concept. First,some building material (e.g., extracellular matrix or biodegradablepolymer) is shaped as needed (e.g., scaffold), seeded with living cells(e.g., stem cells), and bathed with growth factors. While the cellsmultiply, they fill up the scaffold and grow into a three-dimensionaltissue. Once implanted in the body, the tissue will function as asubstitute for the damaged tissue. As blood vessels attach themselves tothe new tissue, the scaffold dissolves, and the newly grown tissueeventually blends in with its surroundings.

In response to the need for more efficient and effective implantmaterials, extracellular matrix (ECM) and other acellular biomaterialshave been used as therapeutic scaffolds for cell attachment andproliferation and as templates for tissue repair (Schmidt C. and BaierJ., 2000, Biomaterials 21:2215-31). Extracellular matrix is a complexstructural entity surrounding and supporting cells. The extracellularmatrix is found within mammalian tissues and is made up of three majorclasses of biomolecules: structural proteins (e.g., collagen andelastin), specialized proteins (e.g., fibrillin, fibronectin, andlaminin), and proteoglycans (e.g., glycosaminoglycans). Although theexact mechanisms through which ECM facilitates repair or regenerationare not known, the composition and the organization of the componentsare considered to be important factors that influence cell attachment,gene expression patterns, and cell differentiation.

Successful implants include extracellular matrix from decellularizedskin, blood vessels, and submucosal tissue. Abatangelo et al. inInternational Publication No. WO 97/18842 describes the preparation ofskin substitutes in vitro by seeding keratinocytes with extracellularmatrix secreted by bone marrow stem cells partially or completelydifferentiated into a specific connective tissue. Naughton in U.S. Pat.No. 5,830,708 describes methods for soft tissue augmentation byinjecting extracellular matrix proteins secreted from fibroblasts whichwere grown on a three-dimensional framework. Bell in U.S. Pat. No.5,800,537 describes a method for producing tissues for grafting usingfragmented extracellular matrix particulates.

Bone marrow ECM is a part of the microenvironment that supportshematopoietic development in the bone marrow. Bone marrow ECM consistsof accessory cells, cytokines, and extracellular matrix. Theextracellular matrix in the bone marrow is comprised mainly offibronectin, hemonectin, thrombospondin, collagen, laminin andglycosaminoglycans, heparan sulfate, dermantan, chondrotin sulfate (CS),and hyaluronic acid (HA). Recent findings show bone marrow is a sourceof multipotent adult stem cells (Jiang et al., 2002, Exp. Hematol.30:826-904). The ability to interact positively with stem cells isespecially important for improving the healing of tissues, such as butnot limited to epithelium tissues, connective tissues, muscle tissues,and nerve tissues.

Restenosis is a closure, re-narrowing or blockage of a peripheral orcoronary artery at the same site caused by an effort to open an occludedportion of the artery by angioplasty (a balloon procedure to open anobstruction or narrowing of a blood vessel) or stent (an expandable,slotted metal tube, inserted into a vessel) procedure. Restenosis occursin 40-50% of patients have angioplasty and in 20-30% with the use ofstents (Ino T. et al., 1997, Acta. Paediatr. 86:367-71). Restenosis haspreviously been addressed by providing stents which are seeded withendothelial cells that had undergone retrovirus-mediated gene transferfor either bacterial beta-galactosidase or human tissue-type plasminogenactivator (Dichek D. et al. in 1989, Circulation 80:1347-53) or stentscoated with antiplatelet agents, anticoagulant agents, antimicrobialagents, antimetabolic agents (U.S. Pat. No. 5,102,417; InternationalPublication No. WO 90/13332). Given the ability of bone marrow ECM toimprove healing of tissue, the use of bone marrow ECM as a therapy islikely to improve patient outcome for occluded arteries.

The present inventors have surprisingly found that bone marrow ECM canbe isolated, is a rich source of matrix proteins and growth factors, andthat injecting or implanting bone marrow extracellular matrix guidestissue repair and regeneration in injured tissue. It is therefore anobject of the present invention to provide a pharmaceutical composition,a medical device, as well as a tissue regeneration scaffold comprising atherapeutically significant amount of decellularized bone marrowextracellular matrix.

3. SUMMARY OF THE INVENTION

To achieve the aforementioned objectives, we have invented a compositioncomprising decellularized bone marrow extracellular matrix, wherein thebone marrow extracellular matrix has been produced in vivo in an animal,preferably a mammal. The composition can be injectable or implantable.Preferably, the decellularized bone marrow extracellular matrix isprepared so the structure of the bone marrow extracellular matrix ismaintained after it is decellularized.

In particular, the invention relates to methods for preparingdecellularized bone marrow extracellular matrix from a bone marrowsample that has been produced in vivo. The bone marrow may bebiologically, chemically, pharmaceutically, physiologically and/ormechanically modified prior to decellularization (see U.S. applicationSer. No. 10/622,293, filed on even date herewith, which is incorporatedby reference in its entirety for all purposes and identified by attorneydocket number 10177-118).

The bone marrow sample, which comprises an extracellular matrix and anon-extracellular matrix component, is decellularized using acombination of physical, chemical, and biological steps. During thestep(s) of decellularization, the bone marrow sample is processed so atleast some of the non-extracellular matrix component is removed and theremaining extracellular matrix is sterilized. Non-extracellular matrixcomponents include, but is not limited to, cells, cell components,blood, bone spicules, extracellular matrix antigens, cytokines, serum,and fat.

Preferably, the decellularized bone marrow extracellular matrix isfurther treated with enzymes to remove nucleic acids and proteins. Morepreferably, the decellularized bone marrow extracellular matrix isfurther processed to limit generation of new immunological sites and topromote subsequent repopulation of the extracellular matrix with cellsfrom a recipient after implantation of the decellularized bone marrowextracellular matrix into the recipient. The decellularized bone marrowextracellular matrix may also be dried, concentrated, lyophilized, orcryopreserved. Preferably, the decellularized bone marrow extracellularmatrix is suspended in a saline solution as a final product.

The composition comprising decellularized extracellular matrix mayfurther comprise a biological material useful for repairing,regenerating or strengthening tissues. Examples of the biologicalmaterial include, but are not limited to, erythropoietin (Epo), stemcell factor (SCF), vascular endothelial growth factor (VEGF),transforming growth factor (TGF), fibroblast growth factor (FGF),epidermal growth factor (EGF), cartilage growth factor (CGF), nervegrowth factor (NGF), keratinocyte growth factor (KGF), skeletal growthfactor (SGF), osteoblast-derived growth factor (BDGF), hepatocyte growthfactor (HGF), insulin-like growth factor (IGF), cytokine growth factor(CGF), stem cell factor (SCF), platelet-derived growth factor (PDGF),endothelial cell growth supplement (ECGS), colony stimulating factor(CSF), growth differentiation factor (GDF), integrin modulating factor(IMF), calmodulin (CaM), thymidine kinase (TK), tumor necrosis factor(TNF), growth hormone (GH), bone morphogenic proteins (BMP), matrixmetalloproteinase (MMP), tissue inhibitor matrix metalloproteinase(TIMP), interferon, interleukins, cytokines, integrin, collagen,elastin, fibrillins, fibronectin, laminin, glycosaminoglycans,vitronectin, proteoglycans, transferrin, cytotactin, tenascin, andlymphokines.

The invention also relates to the injection or implantation ofdecellularized bone marrow extracellular matrix into a subject,preferably a human, to treat, manage or prevent the occurrence ofdefective, diseased, damaged or ischemic tissues or organs. Thedefective, diseased, damaged or ischemic tissue or organ may result fromor is associated with burns, ulcer, wound, bond fracture, diabetes,psoriasis, arthritis, asthma, cystitis, inflammation, infection,ischemia, restenosis, stricture, atherosclerosis, occlusion, stroke,trauma, infarct, vascular disorders, hemophilia, cancer, organ failure(e.g., heart, kidney, lung, liver, intestine), etc. The decellularizedbone marrow extracellular matrix can be injected or implanted alone orin conjunction with other therapeutically or prophylactically effectiveagents or methods to repair, regenerate or strengthen tissues or organs.Preferably, the injected or implanted decellularized bone marrowextracellular matrix is biocompatible. More preferably, thedecellularized bone marrow extracellular matrix minimizes or avoidsimmune response when injected or implanted into a subject. Mostpreferably, the ECM is sterile and is treated with an antibacterialsubstance.

The invention also relates to a medical device comprising decellularizedbone marrow extracellular matrix. Preferably, the medical device is astent or an artificial heart. The decellularized bone marrowextracellular matrix can be coated, preferably by spraying or rolling,onto a surface of the medical device. The decellularized bone marrowextracellular matrix can also be used to form a structural component ofthe medical device. The decellularized bone marrow extracellular matrixcan be used alone or in combination with a biologically active material,preferably paclitaxel, or other polymers to coat or form the medicaldevice. The medical device is suitable for insertion in to a subject,preferably a human.

The invention further relates to a tissue regeneration scaffoldcomprising decellularized bone marrow extracellular matrix and methodsof making the tissue regeneration scaffold.

4. FIGURE

FIG. 1 is a graph showing the effect of DNase-treatment on the DNA andcollagen contents of bone marrow samples.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to decellularized bone marrowextracellular matrix obtained from a bone marrow sample comprising anextracellular matrix and a non-extracellular matrix component. The bonemarrow sample is decellularized using at least a physical, chemicaland/or biological step(s), wherein at least some of thenon-extracellular matrix component is removed.

Non-extracellular matrix component includes, without limitation, cells,cellular component (e.g., cellular lipids, cellular polysaccharides,cellular debris), antigens, blood, bone spicules, serum, and fat. Inpreferred embodiments, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99% by weight, by volumeor by size of the non-extracellular matrix component is removed.

The remaining decellularized extracellular matrix contains componentswhich serve to attract stem cells to the site and/or provide a substrateand/or soluble factors which will promote the repair, regenerationand/or strengthening of tissues. Preferably, the decellularized bonemarrow extracellular matrix is microstructurally and biochemicallyintact, i.e., retains strength, resiliency, and adhesion properties.

5.1 Decellularized Bone Marrow Extracellular Matrix

5.1.1 Isolation of Bone Marrow

Bone marrow (BM) is a soft, spongy tissue that fills the insides ofbones. Most blood cells, including red blood cells, platelets and somewhite blood cells are produced in the bone marrow and are then releasedinto the bloodstream as they become mature. Stem cells found in the bonemarrow are either part of a non-adherent hematopoietic component or anadherent stromal cell component. The adherent stromal cell componentincludes multipotent adult progenitor cells (MAPCs) and mesenchymal stemcells (MSCs). MAPCs have the ability to differentiate into many celltypes including MSCs, endothelial, epithelial and hematopoietic cells,and MSCs have the ability to self-renew by proliferation and todifferentiate into differentiated stromal cells including theosteogenic, chondrogenic, adipogenic, myogenic and fibroblastic lineages(Van Damme A. et al., 2002, Curr. Gene Ther. 2:195-209; Verfaillie C. etal., 2002, Hematology (Am Soc Hematol Educ Program) 369-91).

Bone marrow represents 3-5% of the total body weight and averages about1,500 grams in adults. The hematopoietic bone marrow is organized aroundthe vasculature of the bone cavity. Bone marrow can be sampledrelatively easily using either a needle aspirate or needle biopsytechnique such as those disclosed in U.S. Pat. Nos. 4,314,565;4,481,946; 4,486,188; 4,513,754; 4,630,616; 4,664,128; 5,012,818;5,257,632; and 5,331,972, all of which are incorporated by referenceherein in their entireties.

In a bone marrow aspiration, a special needle (usually a University ofIllinois needle) is inserted beneath the skin and rotated until itpenetrates the cortex, or outer covering of the bone. A small amount ofmarrow is suctioned out of the bone by a syringe attached to the needle.

Bone marrow biopsy may be performed immediately before or after bonemarrow aspiration. The procedure is performed with a special needle,which has a hollow core, much like a leather punch. In bone marrowbiopsy, the needle is inserted, rotated to the right, then to the left,withdrawn, and reinserted at a different angle. This procedure isrepeated until a small chip is separated from the bone marrow.

Generally, aspiration withdraws a fluid specimen (0.5 to 500 ml) thatcontains suspended marrow pustule from the bone marrow and needle biopsyremoves a core of actual marrow cells (not fluid). More information onthe procedures required to perform and evaluate needle aspiration andbiopsy of bone marrow can be found in Hodges A. and Koury M., 1996,Clin. Lab Sci. 9:349-53, the content of which is incorporated byreference herein in its entirety.

Bone marrow aspirations and/or bone marrow biopsies can be performed inphysicians' offices, outpatient clinics and/or hospital settings. Bonemarrow aspirations and/or biopsies should be carried out by trainedindividuals who are aware of the indications, contraindications, andhazards of the procedure. The operators should follow a standardoperating procedure and make an adequate assessment beforehand ofclinical and hematological features (e.g., temperature, heart rate,respiration, and blood pressure). For the patient's comfort and safety,the posterior iliac crest is generally the preferred site of aspiration.Other common sites include the pelvic bone or the breastbone. The siteof puncture should be cleansed first with an antiseptic solution, andthe subject given a local anesthetic.

Bone marrow may also be obtained after a terminal procedure. In thiscase, all marrow is removed from the bones. This may be accomplishedwith the bones in place or removed from the subject. Sterile surgical orlaboratory tools are used to separate the spongy bone containing themarrow from the outer cortical bone.

In preferred embodiments, the bone marrow sample is isolated from amammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, ratsetc.) or a primate (e.g., monkey and human), most preferably a human. Incertain preferred embodiments, the bone marrow sample can be obtainedfrom marrow which has been modified (e.g., manipulation of cells in bonemarrow to alter the ECM produced by host cells). In specificembodiments, the bone marrow sample is obtained from a bone that hasbeen conditioned to produce an amount of a biological material that isin an amount different than that produced by a bone absent theconditioning. The bone may be conditioned before or after the bonemarrow is harvested.

5.1.2 Decellularization Procedures

Decellularization generally refers to the removal of non-extracellularmatrix components, i.e., cells, cellular components, and othernon-extracellular matrix components (e.g., blood, antigens, serum, fat)while leaving intact an extracellular matrix component. Preferably, allor substantially all of the cells, cellular components, andnon-extracellular components are removed. The macroscopic structure ofthe ECM does not need to be maintained. However, in preferredembodiments, the microstructure of the extracellular matrix is retainedafter the bone marrow extracellular matrix is decellularized.

It is believed that the process of decellularization can reduce oreliminate the immune response associated with the cells as well as thecellular components. Acellular vascular tissues have been suggested tobe ideal natural biomaterials for tissue repair and engineering (SchmidtC. and Baier J., supra). Bell in U.S. Pat. No. 5,800,537 describes amethod for producing extracellular matrix particulates first by manuallyand mechanically mincing a tissue source, second by removing the cellremnants from the extracellular matrix of the tissue source in theabsence of a high salt solution, and finally by fragmenting theprocessed extracellular matrix.

A bone marrow sample consists of cells, blood, bone spicules, fat, andextracellular matrix. The bone marrow sample may be decellularized usingone or more physical, chemical and/or biological techniques to removethe cells, blood, bone spicules, and fat (see, e.g. U.S. Pat. Nos.5,192,312; 5,613,982; 5,632,778; 5,843,182; and 5,899,936, which areincorporated herein by reference in their entireties). The physical,chemical and/or biological methods are optimized to preserve as much aspossible the composition of biologically active materials and moreimportantly, the microstructure of the extracellular matrix. Thephysical, chemical and/or biochemical techniques are to be applied in anappropriate order to purify the extracellular matrix component. Thetechniques described herein are preferably performed under asepticconditions.

In certain embodiments, physical forces are used to decellularize thebone marrow samples. For example, vapor phase freezing (slow rate oftemperature decline) and liquid phase freezing (rapid rate oftemperature decline) can cause the formation of intracellular ice thatdisrupts the cells. Colloid-forming materials may be added duringfreeze-thaw cycles to alter ice formation patterns in the samples.Polyvinylpyrrolidone (10% w/v) and dialyzed hydroxyethyl starch (10%w/v) may be added to standard cryopreservation solutions (DMEM, 10%DMSO, 10% fetal bovine serum) to reduce extracellular ice formationwhile permitting formation of intracellular ice (see U.S. Pat. No.5,613,982). The ruptured cell and resulting cellular debris can beremoved by any one or more of the physical, chemical and/or biologicaltechniques described herein.

Similarly, extracellular matrix may be isolated from bone marrow samplesusing centrifugation. For example, a bone marrow sample is centrifugedwith a liquid preparation, preferably Histopaque and more preferablywater or saline, which physically separates components of differentdensities. In a preferred embodiment, the speed for centrifugationranges from 100 to 10,000 g, and more preferably, from 2,500 to 7,500 g,for between 5 to 20 minutes.

Alternatively, extracellular matrix may be isolated from bone marrowsamples using filtration. For example, a bone marrow sample may befiltered using filters of specific pore size. In a specific embodiment,the filter is of a pore size, preferably of 70 to 250 μm, that allowsthe extracellular matrix to pass through. In another specificembodiment, the filter is of a pore size, preferably of 0.1 to 100 μm,that retains the extracellular matrix and larger components. Filtrationis carried out in one step or a series of steps.

It has been reported that modification of the magnitude of the membranedipole potential using compounds such as cholesterol, phloretin, and6-ketocholestanol may also influence binding capacity and disruptsmembrane domains. (Asawakarn T. et al., 2001, J. Biol. Chem.276:38457-63). Accordingly, the present invention further relates tomethods for physically decellularizing bone marrow sample by agitatingcellular membrane potential using electrical (e.g., voltage) means.

Other physical techniques that can be used to decellularize a bonemarrow sample include, but are not limited to, mincing, rinsing,agitation, sedimentation, dialysis, cutting, grinding, pressing,electrical stimulation, electromagnetic forces, hydrostatic orhydrodynamic forces, blasting with sound waves, and ultrasonication.

In certain other embodiments, bone marrow samples may be decellularizedusing at least a chemical techinque. In one embodiment, a bone marrowsample is treated with a solution effective to lyse native cells.Preferably, the solution may be an aqueous hypotonic or low ionicstrength solution formulated to effectively lyse the native tissuecells. Such an aqueous hypotonic solution may be de-ionized water or anaqueous hypotonic buffer. The hypotonic lysis solution may include abuffered solution of water, pH 5.5 to 8, preferably pH 7 to 8. Inpreferred embodiments, the hypotonic lysis solution is free from calciumand zinc ions. Control of the temperature and time parameters during thetreatment of the body tissue with the hypotonic lysis solution, may alsobe employed to limit the activity of proteases. Preferably, the aqueoushypotonic buffer may contain additives that provide suboptimalconditions for the activity of selected proteases, e.g., collagenase,which may be released as a result of cellular lysis. Additives such asmetal ion chelators, e.g., 1,10-phenanthroline andethylenediaminetetraacetic acid (EDTA), create an environmentunfavorable to many proteolytic enzymes.

Accordingly, in preferred embodiments, the bone marrow sample is treatedwith protease inhibitors. General inhibitor solutions manufactured bySigma and Genotech are preferred. Specifically,4-(2-aminoethyl)-benzene-sulfonyl fluoride, E-64, bestatin, leopeptin,aprotin, PMSF, Na EDTA, TIMPs, pepstatin A, phosphoramidon, and1,10-phenanthroline are non-limiting examples of preferred proteaseinhibitors.

In certain other embodiments, the bone marrow sample is treated with adetergent. In a specific embodiment, the bone marrow sample is treatedwith an anionic detergent, preferably sodium dodecyl sulfate in buffer.In another specific embodiment, the bone marrow sample is treated with anon-ionic detergent, such as Triton X-100 or 1% octyl phenoxylpolyethoxyethanol, to solubilize cell membranes and fat. In a preferredembodiment, the bone marrow sample is treated with a combination ofdifferent classes of detergents, for example, a nonionic detergent,Triton X-100, and an anionic detergent, sodium dodecyl sulfate, todisrupt cell membranes and aid in the removal of cellular debris.

Steps should be taken to eliminate any residual detergent levels in theextracellular matrix, so as to avoid interference with the latter'sability to repair, regenerate or strengthen defective, diseased, damagedor ischemic tissues or organs. Selection of detergent type andconcentration will be based partly on its preservation of the structure,composition, and biological activity of the extracellular matrix.

Extracellular matrix may also be isolated from a bone marrow sampleusing at least a biological technique. Preferably, the enzyme treatmentlimits the generation of new immunological sites. For instance, extendedexposure of the bone marrow sample to proteases such as trypsin resultin cell death. However, because at least a portion of the type Icollagen molecule is sensitive to a variety of proteases, includingtrypsin, this may not be the approach of choice for collagenous graftsintended for implant in high mechanical stress locations.

In one embodiment, the bone marrow sample is treated with nucleases toremove DNA and RNA. Nucleases are effective to inhibit cellularmetabolism, protein production, and cell division without degrading theunderlying collagen matrix. Nucleases that can be used for digestion ofnative cell DNA and RNA include both exonucleases and endonucleases. Awide variety of which are suitable for use in this step of the processand are commercially available. For example, exonucleases thateffectively inhibit cellular activity include DNase I and RNase A (SIGMAChemical Company, St. Louis, Mo.) and endonucleases that effectivelyinhibit cellular activity include EcOR I (SIGMA Chemical Company, St.Louis, Mo.) and Hind III (SIGMA Chemical Company, St. Louis, Mo.). It ispreferable that the selected nucleases are applied in a physiologicalbuffer solution which contains ions, such as magnesium and calciumsalts, which are optimal for the activity of the nuclease. It is alsopreferred that the ionic concentration of the buffered solution, thetreatment temperature, and the length of treatment are selected toassure the desired level of effective nuclease activity. The buffer ispreferably hypotonic to promote access of the nucleases to the cellinteriors.

Other enzymatic digestion may be suitable for use herein, for example,enzymes that disrupt the function of native cells in the bone marrowsample may be used. For example, phospholipase, particularlyphospholipases A or C, in a buffered solution, may be used to inhibitcellular function by disrupting cellular membranes of endogenous cells.Preferably, the enzyme employed should not have a detrimental effect onthe extracellular matrix protein. The enzymes suitable for use may alsobe selected with respect to inhibition of cellular integrity, and alsoinclude enzymes which may interfere with cellular protein production.The pH of the vehicle, as well as the composition of the vehicle, willalso be adjusted with respect to the pH activity profile of the enzymechosen for use. Moreover, the temperature applied during application ofthe enzyme to the tissue should be adjusted in order to optimizeenzymatic activity.

In another embodiment, the bone marrow sample is treated so the cellsare removed using immunomagnetic bead separation techniques directed tocell surface markers (e.g., integrins, lineage markers, stem cellmarkers). Immunomagnetic separation (IMS) technology can isolate strainspossessing specific and characteristic surface antigens (Olsvik O. etal., 1994, Clin. Microbiol Rev. 7:43-54). Commercially availableimmunomagnetic separation processes such as Cell Release™ (SigrisResearch, Brea, Calif.) was developed to address the need for a fast,general-purpose way to detach intact cells from beads afterimmunomagnetic separation.

Subsequent to decellularization protocols, the resultant extracellularmatrix is washed at least once with suitable chemical solutions, such assaline, protease, enzymes, detergents, alcohols, acidic or basicsolutions, salt solutions, EDTA, etc., to assure removal of cell debriswhich may include cellular protein, cellular lipids, and cellularnucleic acid, as well as any extracellular debris such as lipids andproteoglycans. Removal of the cellular and extracellular debris reducesthe likelihood of the extracellular matrix eliciting an adverse immuneresponse from the recipient upon injection or implantation. For example,the tissue may be incubated in a balanced salt solution such as Hanks'Balanced Salt Solution (HBSS), preferably sterile. The washing processmay include incubation at a temperature of between about 2° C. and 42°C., with 4° C. to 25° C. most preferable. The transplant tissue matrixmay be incubated in the balanced salt wash solution for up to a day, 3days, 5 days, a week, two weeks, or a month, with changes in washsolution every second or third day. The composition of the balanced saltsolution wash, and the conditions under which it is applied to thetransplant tissue matrix may be selected to diminish or eliminate theactivity of the nuclease or other enzyme utilized during thedecellularization process.

Optionally, an antibacterial, an antifingal or a sterilant or acombination thereof, may be included in the balanced salt wash solutionto protect the decellularized bone marrow extracellular matrix fromcontamination with environmental pathogens. In certain embodiments, thedecellularized bone marrow ECM is sterilized by irradiation, ultravioletlight exposure, ethanol incubation (70-100%), treatment withglutaraldehyde, peracetic acid (0.1-1% in 4% ethanol), chloroform(0.5%), or antimycotic and antibacterial substances.

The decellularized bone marrow extracellular matrix prepared inaccordance with the above is preferably free or substantially free ofits native cells, and additionally, cellular and extra-cellular antigencomponents have been washed out of the extracellular matrix. Preferably,the decellularized bone marrow has been treated in a manner which limitsthe generation of new immunological sites in the collagen matrix. TheECM is obtained as a slurry of small particles. This slurry, includingthe slurry solution, may eventually be processed into an implant.

In addition, the decellularized extracellular matrix may contain asignificant portion of the original tissue mass retaining physicalproperties in regard to strength and elasticity and has components whichare largely collagens but also comprise glycosaminoglycans and proteinsclosely associated with collagen such as the basement membrane complex,laminin and fibronectin.

The above physical, chemical, and biological techniques may also be usedto remove non-extracellular matrix components (e.g., cells, cellularcomponents, blood, bone spicules, extracellular matrix antigens, serum,fat, etc.) prior to removal from the bone marrow cavity. For example,one or more solutions may be perfused through the bone marrow, as blooddoes normally, to solubilize or force non-extracellular matrixcomponents out of the marrow. More specifically, de-ionized water, adetergent solution, an enzymatic solution (e.g., trypsin, dispase,DNase) or EDTA can be forced through the marrow cavity to remove cells,cell debris, blood or fat prior to harvesting the bone marrow samples.

One aspect of the invention further provides the preservation of thedecellularized bone marrow extracellular matrix for later use. Thedecellularized bone marrow extracellular matrix can be freeze-dried forprolonged storage. Likewise, the decellularized bone marrowextracellular matrix can be air-dried by any known standard techniques.In one embodiment, the decellularized bone marrow extracellular matrixcan be concentrated or dehydrated and later reconstituted or rehydrated,respectively, before use.

In yet another embodiment, the decellularized bone marrow extracellularmatrix can be lyophilized. The lyophilized ECM may be in the form of animplant which has pores. Characteristics of the pore structure can becontrolled by process parameters. In yet another embodiment, thedecellularized bone marrow extracellular matrix can be formed as a gel.Preferably, the proteins are temporarily and reversibly denatured. Inyet another embodiment, the decellularized bone marrow extracellularmatrix can be precipitated or co-precipitated with other proteins orbiologics.

In certain embodiments, the decellularized bone marrow extracellularmatrix is cryopreserved. For general methods of cryopreservation seeBrockbank K., Basic Principles of Viable Tissue Preservation. In:Transplantation Techniques and Use of Cryopreserverd Allograft CardiacValves and Vascular Tissue. D. R. Clarke (ed.), Adams Publishing Group,Ltd., Boston. pp 9-23, which is hereby incorporated by reference hereinin its entirety. Cryopreservation of decellularized bone marrowextracellular matrix would assure a supply or inventory of substantiallynon-immunogenic extracellular matrices which, upon thawing, would beready for further treatment according to the subsequent steps of thisinvention, or further processed as desired to provide an implant tissueproduct.

In another particular embodiment, the decellularized bone marrowextracellular matrix is treated to enhance the ingrowth and attachmentof the recipient's own cells. Growth factors, cytokines, geneticmaterial, etc., for example, can be used to incubate the decellularizedextracellular matrix prior to its injection or implantation into asubject or prior to its coating on or incorporation into a medicaldevice. Preferred growth factors include, but are not limited to,erythropoietin (Epo), endothelial growth factor (VEGF), transforminggrowth factor (TGF), fibroblast growth factor (FGF), epidermal growthfactor (EGF), cartilage growth factor (CGF), nerve growth factor (NGF),keratinocyte growth factor (KGF), skeletal growth factor (SGF),osteoblast-derived growth factor (BDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), cytokine growth factor (CGF), stemcell factor (SCF), platelet-derived growth factor (PDGF), endothelialcell growth supplement (ECGS), colony stimulating factor (CSF), growthdifferentiation factor (GDF), integrin modulating factor (IMF),calmodulin (CaM), thymidine kinase (TK), growth hormone (GH), bonemorphogenic proteins (BMP) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6(Vgr-1), BMP-7 (PO-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-14,BMP-15, BMP-16), matrix metalloproteinase (MMP), and tissue inhibitormatrix metalloproteinase (TIMP). Preferred cytokines include, but arenot limited to, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15), lymphokines, tumornecrosis factor (TNF) (e.g., TNF-α, TNF-β), and interferons.

5.2 Therapeutic Use

The invention relates generally to the use of decellularized bone marrowextracellular matrix to repair or regenerate defective, diseased,damaged or ischemic tissues or organs in a subject. Pharmaceuticalcompositions, medical devices and tissue regeneration scaffoldscomprising decellularized bone marrow extracellular matrix can beinjected or implanted into a subject in need thereof.

In certain embodiments, the decellularized bone marrow extracellularmatrix may be used to treat defective, diseased, damaged or ischemictissues or organs which include, but are not limited to, head, neck,eye, mouth, throat, esophagus, chest, bone, ligament, cartilage,tendons, lung, colon, rectum, stomach, prostate, breast, ovaries,fallopian tubes, uterus, cervix, testicles or other reproductive organs,hair follicles, skin, diaphragm, thyroid, blood, muscles, bone marrow,heart, lymph nodes, blood vessels, large intestine, small intestine,kidney, liver, pancreas, brain, spinal cord, and the central nervoussystem.

In particular, the decellularized bone marrow extracellular matrix maybe used to treat diseases that may benefit from improved angiogenesis,cell proliferation and tissue regeneration. Such diseases or conditionsinclude, but are not limited to, burns, ulcer, wound, bone fracture,diabetes, psoriasis, arthritis, asthma, cystitis, inflammation,infection, ischemia, restenosis, stricture, atherosclerosis, occlusion,stroke, trauma, infarct, vascular disorders, hemophilia, cancer, andorgan failure (e.g., heart, kidney, lung, liver, intestine, etc.).

In a specific embodiment, the present invention regenerates or replacesat least 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, at least 10%, at least 5%, or at least 1% of defective,diseased, damaged or ischemic cells from the affected tissue or organ.

The methods of the present invention is provided for an animal,including but not limited to mammals such as a non-primate (e.g., cows,pigs, horses, chickens, cats, dogs, rats, etc.), and a primate (e.g.,monkey such as acynomolgous monkey and a human). In a preferredembodiment, the subject is a human.

The decellularized bone marrow extracellular matrix is useful alone orin combination with other treatment modalities. In certain embodiments,the treatment of the present invention further includes theadministration of one or more immunotherapeutic agents, such asantibodies and immunomodulators, which include, but are not limited to,HERCEPTIN®, RITUXAN®, OVAREX™, PANOREX®, BEC2, IMC-C225, VITAXIN™,CAMPATH® I/H, Smart M195, LYMPHOCIDE™, Smart I D10, ONCOLYM™, rituximab,gemtuzumab, or trastuzumab. In certain other embodiments, the treatmentmethod further comprises hormonal treatment. Hormonal therapeutictreatments comprise hormonal agonists, hormonal antagonists (e.g.,flutamide, tamoxifen, leuprolide acetate (LUPRON™), LH-RH antagonists),inhibitors of hormone biosynthesis and processing, steroids (e.g.,dexamethasone, retinoids, betamethasone, cortisol, cortisone,prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids,estrogen, testosterone, progestins), antigestagens (e.g., mifepristone,onapristone), and antiandrogens (e.g., cyproterone acetate).

5.2.1 Pharmaceutical Compositions

The decellularized bone marrow extracellular matrix can be formulatedinto pharmaceutical compositions that are suitable for administration toa subject. Such compositions comprise a prophylactically ortherapeutically effective amount of the decellularized bone marrowextracellular matrix as disclosed herein, and a pharmaceuticallyacceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete) or, more preferably, MF59C.1 adjuvantavailable from Chiron, Emeryville, Calif.), excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Other examples of suitable pharmaceutical vehicles aredescribed in “Remington: the Science and Practice of Pharmacy”, 20thed., by Mack Publishing Co. 2000.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed from an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Various delivery systems are known and can be used to administer thecompositions of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. Methods ofadministering a prophylactic or therapeutic amount of the compositionsof the invention include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intraperitoneal,intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal,inhaled, and oral routes). Preferably, a prophylactic or therapeuticamount of the compositions of the invention is directly injected at thedefective, diseased, damaged or ischemic tissue. The compositioncomprising decellularized extracellular matrix of conditioned bodytissue may be administered by any convenient route, for example, byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents, preferably paclitaxel. Administration can be systemic or local.In addition, it may be desirable to introduce the pharmaceuticalcomposition of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

In another embodiment, the decellularized bone marrow extracellularmatrix can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, 1990, Science 249:1527-1533; Sefton,1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). Any techniqueknown to one of skill in the art can be used to produce sustainedrelease formulations comprising the decellularized extracellular matrixof the invention. See, e.g., U.S. Pat. No. 4,526,938; InternationalPublication Nos. WO 91/05548 and WO 96/20698; Ning et al., 1996,Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA Journal ofPharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, Pro.Int'l. Symp. Control Rel. Bioact. Mater. 24:853-854; and Lam et al.,1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each ofwhich is incorporated herein by reference in its entirety.

In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the decellularized extracellularmatrix material (see, e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105); U.S. Pat. Nos. 5,679,377; 5,916,597;5,912,015; 5,989,463; and 5,128,326; International Publication Nos. WO99/15154 and WO 99/20253). Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glucosides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable during storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity to the target, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, 1984, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138).

The amount of the pharmaceutical composition which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays and animal models mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

5.2.2 Medical Devices

The decellularized bone marrow extracellular matrix of the invention mayalso be used to form a medical or prosthetic device, preferably a stentor an artificial heart, which may be implanted in the subject. Morespecifically, the decellularized bone marrow extracellular matrix of theinvention may be incorporated into the base material needed to make thedevice. For example, in stent comprising a sidewall of elongated membersor wire-like elements, the decellularized bone marrow extracellularmatrix can be used to form the elongated members or wire-like elements.On the other hand, the decellularized bone marrow extracellular matrixcan be used to coat or cover the medical device.

The medical devices of the present invention may be inserted orimplanted into the body of a patient.

5.2.2.1 Types of Medical Device

Medical devices that are useful in the present invention can be made ofany biocompatible material suitable for medical devices in general whichinclude without limitation natural polymers, synthetic polymers,ceramics and metallics. Metallic material is more preferable. Suitablemetallic materials include metals and alloys based on titanium (such asnitinol, nickel titanium alloys, thermo-memory alloy materials),stainless steel, tantalum, nickel-chrome, or certain cobalt alloysincluding cobalt-chromium-nickel alloys such as Elgiloy® and Phynox®.Metallic materials also include clad composite filaments, such as thosedisclosed in WO 94/16646.

Metallic materials may be made into elongated members or wire-likeelements and then woven to form a network of metal mesh. Polymerfilaments may also be used together with the metallic elongated membersor wire-like elements to form a network mesh. If the network is made ofmetal, the intersection may be welded, twisted, bent, glued, tied (withsuture), heat sealed to one another; or connected in any manner known inthe art.

The polymer(s) useful for forming the medical device should be ones thatare biocompatible and avoid irritation to body tissue. They can beeither biostable or bioabsorbable. Suitable polymeric materials includewithout limitation polyurethane and its copolymers, silicone and itscopolymers, ethylene vinyl-acetate, polyethylene terephtalate,thermoplastic elastomers, polyvinyl chloride, polyolefins, cellulosics,polyamides, polyesters, polysulfones, polytetrafluorethylenes,polycarbonates, acrylonitrile butadiene styrene copolymers, acrylics,polylactic acid, polyglycolic acid, polycaprolactone, polylacticacid-polyethylene oxide copolymers, cellulose, collagens, and chitins.

Other polymers that are useful as materials for medical devices includewithout limitation dacron polyester, poly(ethylene terephthalate),polycarbonate, polymethylmethacrylate, polypropylene, polyalkyleneoxalates, polyvinylchloride, polyurethanes, polysiloxanes, nylons,poly(dimethyl siloxane), polycyanoacrylates, polyphosphazenes,poly(amino acids), ethylene glycol I dimethacrylate, poly(methylmethacrylate), poly(2-hydroxyethyl methacrylate),polytetrafluoroethylene poly(HEMA), polyhydroxyalkanoates,polytetrafluorethylene, polycarbonate, poly(glycolide-lactide)co-polymer, polylactic acid, poly(ε-caprolactone),poly(β-hydroxybutyrate), polydioxanone, poly(γ-ethyl glutamate),polyiminocarbonates, poly(ortho ester), polyanhydrides, alginate,dextran, chitin, cotton, polyglycolic acid, polyurethane, or derivatizedversions thereof, i.e., polymers which have been modified to include,for example, attachment sites or cross-linking groups, e.g., RGD, inwhich the polymers retain their structural integrity while allowing forattachment of cells and molecules, such as proteins, nucleic acids, andthe like.

Furthermore, although the invention can be practiced by using a singletype of polymer to form the medical device, various combinations ofpolymers can be employed. The appropriate mixture of polymers can becoordinated to produce desired effects when incorporated into a medicaldevice. In certain preferred embodiments, the decellularizedextracellular matrix is mixed with a polymer.

The decellularized bone marrow extracellular matrix of the invention mayalso be used alone or in combination with a polymer described above toform the medical device such as through formation of a compositematerial. The decellularized bone marrow extracellular matrix may bedried to increase its mechanical strength. The dried decellularized bonemarrow extracellular matrix may then be used as the base material toform a whole or part of the medical device. In preferred embodiments,the decellularized bone marrow extracellular matrix constitutes at least5%, at least 10%, at least 25%, at least 50%, at least 80%, at least90%, at least 95%, at least 99% by weignt or by size of the medicaldevice.

Examples of the medical devices suitable for the present inventioninclude, but are not limited to, stents, surgical staples, catheters(e.g., central venous catheters and arterial catheters), guidewires,cannulas, cardiac pacemaker leads or lead tips, cardiac defibrillatorleads or lead tips, implantable vascular access ports, blood storagebags, blood tubing, vascular or other grafts, intra-aortic balloonpumps, heart valves, cardiovascular sutures, total artificial hearts andventricular assist pumps, and extra-corporeal devices such as bloodoxygenators, blood filters, hemodialysis units, hemoperfusion units andplasmapheresis units.

Medical devices of the present invention include those that have atubular or cylindrical-like portion. The tubular portion of the medicaldevice need not to be completely cylindrical. For instance, thecross-section of the tubular portion can be any shape, such asrectangle, a triangle, etc., not just a circle. Such devices include,without limitation, stents and grafts. A bifurcated stent is alsoincluded among the medical devices which can be fabricated by the methodof the present invention.

Medical devices which are particularly suitable for the presentinvention include any kind of stent for medical purposes which is knownto the skilled artisan. Suitable stents include, for example, vascularstents such as self-expanding stents and balloon expandable stents.Examples of self-expanding stents useful in the present invention areillustrated in U.S. Pat. Nos. 4,655,771 and 4,954,126 issued to Wallstenand U.S. Pat. No. 5,061,275 issued to Wallsten et al. Examples ofappropriate balloon-expandable stents are shown in U.S. Pat. No.5,449,373 issued to Pinchasik et al.

5.2.2.2 Methods of Coatine the Medical Device

In the present invention, the decellularized bone marrow extracellularmatrix of the invention, preferably in combination with a biologicallyactive material such as paclitaxel, can be applied by any method to asurface of a medical device to form a coating. Examples of suitablemethods are spraying, brushing, swabbing, dipping, rolling,electrostatic deposition and all modern chemical ways of immobilizationof bio-molecules to surfaces. Preferably, the decellularized bone marrowextracellular matrix is applied to a surface of a medical device byspraying and rolling. In one embodiment of the present invention, morethan one coating method can be used to make a medical device. In certainembodiments, the decellularized extracellular matrix is placed into acarrier in order to apply it to the device surface. Non-limitingexamples of carriers include SIBS, PLGA, PGA, collagen, etc.

Furthermore, before applying the coating composition, the surface of themedical device is optionally subjected to a pre-treatment, such asroughening, oxidizing, sputtering, plasma-deposition or priming inembodiments where the surface to be coated does not comprisedepressions. Sputtering is a deposition of atoms on the surface byremoving the atom from the cathode by positive ion bombardment through agas discharge. Also, exposing the surface of the device to a primer is apossible method of pre-treatment.

Coating compositions suitable for applying coating materials to thedevices of the present invention can include a polymeric material andpreferably a biologically active material dispersed or dissolved in asolvent suitable for the medical device, which are known to the skilledartisan. The solvents used to prepare coating compositions include oneswhich can dissolve the polymeric material into solution or suspend thepolymeric material and do not alter or adversely impact the therapeuticproperties of the biologically active material employed. For example,useful solvents for silicone include tetrahydrofuran (THF), chloroform,toluene, acetone, isooctane, 1,1,1-trichloroethane, dichloromethane, andmixture thereof.

The polymeric material should be a material that is biocompatible andavoids irritation to body tissue. Preferably the polymeric materialsused in the coating composition of the present invention are selectedfrom the following: polyurethanes, silicones (e.g., polysiloxanes andsubstituted polysiloxanes), and polyesters. Also preferable as apolymeric material is styrene-isobutylene-styrene (SIBS). Other polymerswhich can be used include ones that can be dissolved and cured orpolymerized on the medical device or polymers having relatively lowmelting points that can be blended with biologically active materials.Additional suitable polymers include, thermoplastic elastomers ingeneral, polyolefins, polyisobutylene, ethylene-alphaolefin copolymers,acrylic polymers and copolymers, vinyl halide polymers and copolymerssuch as polyvinyl chloride, polyvinyl ethers such as polyvinyl methylether, polyvinylidene halides such as polyvinylidene fluoride andpolyvinylidene chloride, polyacrylonitrile, polyvinyl ketones, polyvinylaromatics such as polystyrene, polyvinyl esters such as polyvinylacetate, copolymers of vinyl monomers, copolymers of vinyl monomers andolefins such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS (acrylonitrile-butadiene-styrene)resins, ethylene-vinyl acetate copolymers, polyamides such as Nylon 66and polycaprolactone, alkyd resins, polycarbonates, polyoxymethylenes,polyimides, polyethers, epoxy resins, rayon-triacetate, cellulose,cellulose acetate, cellulose butyrate, cellulose acetate butyrate,cellophane, cellulose nitrate, cellulose propionate, cellulose ethers,carboxymethyl cellulose, collagens, chitins, polylactic acid,polyglycolic acid, polylactic acid-polyethylene oxide copolymers, EPDM(etylene-propylene-diene) rubbers, fluorosilicones, polyethylene glycol,polysaccharides, phospholipids, and combinations of the foregoing.

More preferably for medical devices which undergo mechanical challenges,e.g. expansion and contraction, the polymeric materials should beselected from elastomeric polymers such as silicones (e.g. polysiloxanesand substituted polysiloxanes), polyurethanes, thermoplastic elastomers,ethylene vinyl acetate copolymers, polyolefin elastomers, and EPDMrubbers. Because of the elastic nature of these polymers, the coatingcomposition is capable of undergoing deformation under the yield pointwhen the device is subjected to forces, stress or mechanical challenge.

The term “biologically active material” encompasses therapeutic agents,such as drugs, and also genetic materials and biological materials. Thegenetic materials mean DNA or RNA, including, without limitation, ofDNA/RNA encoding a useful protein stated below, intended to be insertedinto a human body including viral vectors and non-viral vectors. Thebiological materials include cells, yeasts, bacteria, proteins,peptides, cytokines and hormones. Examples for peptides and proteinsinclude vascular endothelial growth factor (VEGF), transforming growthfactor (TGF), fibroblast growth factor (FGF), epidermal growth factor(EGF), cartilage growth factor (CGF), nerve growth factor (NGF),keratinocyte growth factor (KGF), skeletal growth factor (SGF),osteoblast-derived growth factor (BDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), cytokine growth factor (CGF), stemcell factor (SCF), platelet-derived growth factor (PDGF), endothelialcell growth supplement (ECGS), granulocyte macrophage colony stimulatingfactor (GM-CSF), growth differentiation factor (GDF), integrinmodulating factor (IMF), calmodulin (CaM), thymidine kinase (TK), tumornecrosis factor (TNF), growth hormone (GH), bone morphogenic protein(BMP) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (PO-1),BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-14, BMP-15, BMP-16, etc.),matrix metalloproteinase (MMP), tissue inhibitor matrixmetalloproteinase (TIMP), cytokines, interleukin (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15,etc.), lymphokines, interferon, integrin, collagen, elastin, fibrillins,fibronectin, vitronectin, laminin, glycosaminoglycans, proteoglycans,transferrin, cytotactin, and tenascin. Currently preferred BMP's areBMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7. These dimeric proteins can beprovided as homodimers, heterodimers, or combinations thereof, alone ortogether with other molecules. Cells can be of human origin (autologousor allogeneic) or from an animal source (xenogeneic), geneticallyengineered, if desired, to deliver proteins of interest at thetransplant site. The delivery media can be formulated as needed tomaintain cell function and viability. Cells include progenitor cells(e.g., endothelial progenitor cells), stem cells (e.g., mesenchymal,hematopoietic, neuronal), stromal cells, parenchymal cells,undifferentiated cells, fibroblasts, macrophage, and satellite cells.Biologically active materials also include non-genetic therapeuticagents, such as:

-   anti-thrombogenic agents such as heparin, heparin derivatives,    urokinase, and PPack (dextrophenylalanine proline arginine    chloromethylketone);-   anti-proliferative agents such as enoxaprin, angiopeptin, or    monoclonal antibodies capable of blocking smooth muscle cell    proliferation, hirudin, and acetylsalicylic acid, amlodipine and    doxazosin;-   anti-inflammatory agents such as glucocorticoids, betamethasone,    dexamethasone, prednisolone, corticosterone, budesonide, estrogen,    sulfasalazine, and mesalamine;-   antineoplastic/antiproliferative/anti-miotic agents such as    paclitaxel, 5-fluorouracil, cisplatin, vinblastine, cladribine,    vincristine, epothilones, methotrexate, azathioprine, adriamycin and    mutamycin; endostatin, angiostatin and thymidine kinase inhibitors,    taxol and its analogs or derivatives;-   anesthetic agents such as lidocaine, bupivacaine, and ropivacaine;-   anti-coagulants such as D-Phe-Pro-Arg chloromethyl keton, an RGD    peptide-containing compound, heparin, antithrombin compounds,    platelet receptor antagonists, anti-thrombin antibodies,    anti-platelet receptor antibodies, aspirin (aspirin is also    classified as an analgesic, antipyretic and anti-inflammatory drug),    dipyridamole, protamine, hirudin, prostaglandin inhibitors, platelet    inhibitors and tick antiplatelet peptides;-   DNA demethylating drugs such as 5-azacytidine, which is also    categorized as a RNA or DNA metabolite that inhibit cell growth and    induce apoptosis in certain cancer cells;-   vascular cell growth promoters such as growth factors, vascular    endothelial growth factors (VEGF, all types including VEGF-2),    growth factor receptors, transcriptional activators, and    translational promoters;-   vascular cell growth inhibitors such as antiproliferative agents,    growth factor inhibitors, growth factor receptor antagonists,    transcriptional repressors, translational repressors, replication    inhibitors, inhibitory antibodies, antibodies directed against    growth factors, bifunctional molecules consisting of a growth factor    and a cytotoxin, bifunctional molecules consisting of an antibody    and a cytotoxin;-   cholesterol-lowering agents; vasodilating agents; and agents which    interfere with endogenous vasoactive mechanisms;-   anti-oxidants, such as probucol;-   antibiotic agents, such as penicillin, cefoxitin, oxacillin,    tobranycin, rapamycin;-   angiogenic substances, such as acidic and basic fibroblast growth    factors, estrogen including estradiol (E2), estriol (E3) and 17-Beta    Estradiol;-   drugs for heart failure, such as digoxin, beta-blockers,    angiotensin-converting enzyme (ACE) inhibitors including captopril,    enalopril, and statins and related compounds; and

In certain embodiments, the medical device of the present invention iscovered with one coating layer. In certain other embodiments, themedical device of the present invention is covered with more than onecoating layer. In preferred embodiments, the medical device is coveredwith different coating layers. For example, the coating can comprise afirst layer and a second layer that contain different biologicallyactive materials. Alternatively, the first layer and the second layermay contain an identical biologically active material having differentconcentrations. In one embodiment, either the first layer or the secondlayer may be free of biologically active material.

5.2.3 Tissue Regeneration Scaffold

One aspect of the invention provides for use alone or through theincorporation of the decellularized bone marrow extracellular matrixinto a biocompatible material for implantation into a subject,preferably human. In a preferred embodiment, the biocompatible materialis in the form of a scaffold.

The scaffold may be of decellularized bone marrow ECM or combined withnatural collagen or synthetic polymer. These scaffolds may be producedby freeze-drying, air-drying, molding or salt leaching. In certainpreferred embodiments, the scaffold serves as a template for cellproliferation and ultimately tissue formation. In a specific embodiment,the scaffold allows the slow release of the decellularized bone marrowextracellular matrix into the surrounding tissue. As the cells in thesurrounding tissue begin to multiply, they fill up the scaffold and growinto three-dimensional tissue. Blood vessels then attach themselves tothe newly grown tissue, the scaffold dissolves, and the newly growntissue eventually blends in with its surrounding.

5.2.4 Cell Culture Substrate

Certain cell types require a substrate other than culture plastic togrow and retain proper function outside their native environment (e.g.,embryonic stem cells, some neural cells). Surfaces coated withextracellular matrix proteins (e.g., laminin, fibronectin, collagen,Matrigel) have been shown to enhance the growth of certain cell typescompared to the uncoated surface. Also, cells grown on top of or withinthree-dimensional gels or scaffolds grow and function differently thanthose on two-dimensional coated or uncoated surfaces.

In certain embodiments, the decellularized bone marrow extracellularmatrix of the invention is used for cell culture. In one embodiment,some or all of the decellularized bone marrow extracellular matrix canbe solubilized in an acidic solution, preferably 0.05 to 0.5 M aceticacid, with or without an enzyme which breaks apart the collagen molecule(e.g., pepsin, trypsin, dispase, collagenase). The resulting solutioncontaining preferably 0.5 to 15 mg/ml of decellularized bone marrowextracellular matrix will gel when the pH is neutralized (temperaturepreferably between 25° C. to 40° C.). During the gelation process thesolution can be added to a container to coat the exposed surfaces,seeded with cells to form a gel containing cells, or cast into sheet orslabs.

In another embodiment, a suspension of decellularized bone marrowextracellular matrix or a solution, as described above, can be dried toform a sheet of ECM or adsorbed onto a surface. The thickness will bedetermined by the concentration of ECM in the liquid and the volume ofliquid per surface area of the container in which it is held. A glass ornon-stick (e.g., Teflon, silicone) container is preferred to enable theremoval of the sheet without destroying its integrity. Although notnecessary, the sheet can be cross-linked prior to or after removal fromthe container in which it was dried. Examples of cross-linkingtreatments include but are not limited to UV light or other highfrequency photon source, aldehydes, carbodiimides, and severedehydration. Cells may be seeded directly onto the sterilized coatedcontainers or onto the sheets which have been removed.

In yet another embodiment, a suspension of decellularized bone marrowextracellular matrix or a solution, as described above, can befreeze-dried within a container to form a porous coating or layer on thesurface of a culture container. The ratio of freezing and concentrationof ECM in the liquid will determine the pore structure and pore volumefraction. Altering these properties will allow control of thoseparameters. Preferably, the freezing temperature will be between −10° C.and −196° C.

6. EXAMPLES 6.1 Example 1

First, a bone marrow sample is isolated from a pig or cow. Second, thebone marrow sample is filtered to remove bone spicules. Third, theremaining bone marrow sample is centrifuged in saline to remove the fat.Fourth, a non-ionic detergent is added to the remaining bone marrowsample to solubilize the cell membranes and any remaining fat. Theremaining bone marrow sample is then rinsed with saline and filtered toremove the remaining extracellular matrix components. The rinsing stepis repeated at least one time. Further, the remaining bone marrow sampleis further treated with DNase and RNase to breakdown remaining nucleicacids and rinsed with saline to remove the remaining extracellularmatrix components. Finally, the resulting extracellular matrix issterilized using standard techniques in the art, e.g., peracetic acid(0.1%).

6.2 Example 2

First, a bone marrow sample is removed from the bone marrow cavity andthe cortical bone by cutting, scooping, chiseling or sawing. The marrowis then reduced to small pieces by cutting, grinding, chopping,shearing, or crushing. The blood components are removed by rinsing, oneor more times, in buffered saline or water. Next, the bone is separatedfrom the ECM and any other remaining components by vigorous agitation(e.g., vortexing, shaking). After allowing the bone fragments to settleto the bottom, the non-bone components are removed by carefullydecanting or aspirating (e.g., pipet). This process is repeated byadding more saline or water to the remaining bone. The non-bonecomponents are treated with a combination of non-ionic detergent, acidicsolution, alcohol solution, and basic solution to solubilize non-ECMcomponents. Alternatively, the non-blood components are treated with anacidic or enzymatic solution (0.5 M acetic acid with pepsin 1 mg/ml).The solubilized ECM components are then separated by decanted followingcentrifugation and filtering with a 20 to 100 micron filter. Thefiltered solids will contain some of the bone marrow ECM. This ECM isprocessed further for use by lyophilization, precipitation, or gelation.

6.3 Example 3

6.3.1 Isolation Procedure

Ribs and sternum from a freshly killed pig are collected and thecortical bone is removed. Large pieces of bone marrow are obtained bypiercing, cutting and prying at the cortical bone-bone marrow interfacewith a sharp, stout knife. The large pieces of bone marrow aresubsequently reduced in size to less than 5 mm on a side by cutting,crushing or shearing. Blood which remains in these pieces is rinsed awayby multiple rinses using a buffered saline with protease inhibitorProtease-Arrest™ (Genotech, St. Louis, Mo.). For each rinse, the marrowis first placed in the solution and slowly agitated for 30 minutes. Therinsing solution is then removed from the bone marrow pieces with apipet. Following the final rinse, more buffered saline with proteaseinhibitor Protease-Arrest™ is added to the bone marrow piece. The marrowis separated from the bone pieces by vigorous agitation with a standardlab vortex mixer. The resulting suspension of marrow is transferred toanother container using a pipet. This process is repeated several timesuntil the bone is substantially free of marrow.

Finally, the marrow suspension is centrifuged at about 3,000 g for 15minutes and the supernatant is removed. Distilled water with proteaseinhibitor Protease-Arrest™ is added to the pellet and gently agitated tolyse and remove the cells. This suspension is filtered using a 40 micronfilter. The filtered solid is collected and processed for use.Optionally, the distilled water rinse and subsequent filtering step canbe repeated to remove any remaining cells or cell debris. Asepticconditions are maintained throughout the isolation procedure.

6.3.2 Production of a Freeze-Dried Porous Scaffold

0.05 M acetic acid is added to the solid material obtained from theisolation procedure discussed above to yield 1 to 6 mg of bone marrowECM per ml. The mixture is vortexed or homogenized until a suspension isformed. The suspension is transferred to a plastic or metal mold andplaced in a −70° C. freezer for 4 hours or until frozen. Afterwards, themold is transferred to a freeze drier and pull a vacuum of <100 mTorr.The vacuum is held at a temperature between 0° C. and 25° C. for 12hours or until all liquid has sublimated. Optionally, the sample can becross-linked by holding a vacuum of <300 mTorr at 110° C. for 24 hours.Prior to use, the scaffold formed in the mold is rehydrated by placingthe scaffold in sterile saline at room temperature for 12 hours.Preferably, prior to rehydration, the scaffold is sterilized by firstplacing in 1% peracetic acid and 70% ethanol for 12 hours, and thenrinsing three times in sterile saline prior to implantation.

6.4 Example 4

6.4.1 Isolation Procedure

Ribs and sternum from a freshly killed pig were collected and thecortical bone was removed. Large pieces of bone marrow were obtained bypiercing, cutting, and prying at the cortical bone-bone marrow interfacewith a sharp, stout knife. The large pieces of bone marrow weresubsequently reduced in size to less than 5 mm on a side by cutting,crushing or shearing. The bone marrow pieces were washed in a bufferedsaline with protease inhibitor Protease-Arrest™. A DNase solution (10mg/ml DNase solution (Sigma), 5 mM MgCl₂.6H₂O, and 0.5 mM CaCl₂) wasadded to cover the bone marrow pieces. The suspension was gently shakenat 37° C. for 50 minutes. Afterwards, the DNase solution was removed andbuffered saline was added. The suspension was shaken at 4° C. for 10minutes.

The solution was removed and more buffered saline was added. Thesuspension was vortexed until all fibrous material was removed from thebone marrow pieces. A first supernatant was collected. More bufferedsaline was added to rinse the bone marrow pieces and a secondsupernatant was collected.

The first and second supernatant were pooled together and centrifuged at10,000 RPM for 7 minutes. The pellet was resuspended in 1 ml DNasesolution and shaken at 37° C. for 20 minutes. After centrifuging thesuspension at 10,000 RPM for 7 minutes, the supernatant was discarded.Buffered saline was added and the suspension was shaken at 4° C. for 20minutes. After centrifuging the suspension at 10,000 RPM for 7 minutes,the supernatant was again discarded. The steps of adding the bufferedsaline, shaking the suspension at 4° C. for 20 minutes, centrifuging at10,000 RPM for 7 minutes, and discarding the supernatant were repeatedtwice. The suspension was centrifuged at 10,000 RPM for 10 minutes tocollect the pellet.

6.4.2 Experimental Design

The PicoGreen® dsDNA quantification assay (Turner BioSystems) was usedto determine the DNA content of untreated bone marrow samples in thecontrol group and the DNase-treated bone marrow samples in theexperimental group.

The Sircol™ COLLAGEN assay (Biocolor Ltd.) was used to determine thepepsin/acetic acid soluble collagen content of untreated bone marrowsamples in the control group and the DNase-treated bone marrow samplesin the experimental group

6.4.3 Data

As shown in FIG. 1, the average (n=5) DNA content of DNase treatedsamples was 4 times lower (p<0.005) than non-DNase-treated samples andapproaches the lower detection limit for the assay. In contrast, theaverage (n=5) collagen content of DNase treated samples was 1.5 timeshigher than non-DNase-treated samples, although the difference was notsignificant (p=0.18).

Together, the data suggests that the isolation method described supra(Section 6.4.1) retains more collagen and causes the DNA in the bonemarrow sample to be washed away or fragmented. Therefore, this isolationmethod lowers the overall amount of cells or cell debris in theremaining collagen or extracellular matrix.

6.4.4 Production of an Air-Dried Sheet

0.05 acetic acid is added to the solid material obtained from theisolation procedure discussed above to yield 1 to 6 mg of marrow ECM perml. The mixture is vortexted for 5 minutes, ultrasonicated for 2minutes, or homogenized for less than 1 minute until a suspension isformed. The suspension is transferred to a Teflon, silicone, or glassmold (1 mg/ml suspension at 1 ml/10 cm²). Afterwards, the mold istransferred to a dryer and dried under a clean, sterile hood at roomtemperature for 72 hours. Optionally, the sample can be cross-linked byholding a vacuum of <300 mTorr at 110° C. for 24 hours. Prior to use,the scaffold formed in the mold is rehydrated by placing the scaffold insterile saline at room temperature for 12 hours. Preferably, prior torehydration, the scaffold is sterilized by first placing in 1% peraceticacid and 70% ethanol for 12 hours, and then rinsing three times insterile saline prior to implantation.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1. A biocompatible material comprising decellularized bone marrowextracellular matrix, wherein the bone marrow extracellular matrix hasbeen produced in vivo in an animal, and wherein the biocompatiblematerial is in the form of a scaffold.
 2. The biocompatible material ofclaim 1, wherein the decellularized bone marrow extracellular matrix isproduced by a method comprising the steps of: (a) obtaining from asubject a bone marrow sample having an extracellular matrix andnon-extracellular matrix components; (b) processing the bone marrowsample to remove at least some of the non-extracellular matrix componentto obtain decellularized bone marrow extracellular matrix; and (c)sterilizing the decellularized bone marrow extracellular matrix.
 3. Thebiocompatible material of claim 2, wherein the non-extracellular matrixcomponent comprises cells, cell components, antigens, cytokines, blood,bone spicules, serum, and fat.
 4. The biocompatible material of claim 2,wherein at least 50% of the non-extracellular matrix component isremoved.
 5. The biocompatible material of claim 2, wherein at least 80%of the non-extracellular matrix component is removed.
 6. Thebiocompatible material of claim 2, wherein at least 95% of thenon-extracellular matrix component is removed.
 7. The biocompatiblematerial of claim 2, wherein the bone marrow extracellular matrix isarranged in a structure and wherein the structure is maintained afterthe bone marrow extracellular matrix is decellularized.
 8. Thebiocompatible material of claim 2, wherein the method further comprisesthe step of enzymatically treating the decellularized bone marrowextracellular matrix.
 9. The biocompatible material of claim 2, whereinthe method further comprises suspending the decellularized bone marrowextracellular matrix in a saline solution.
 10. The biocompatiblematerial of claim 1, wherein the animal is a mammal.
 11. Thebiocompatible material of claim 1, wherein the mammal is selected fromthe group consisting of cow, pig, horse, chicken, cat, dog, rat, monkey,and human.
 12. The biocompatible material of claim 11, wherein themammal is human, and wherein the human is an adult, adolescent, orneonate.
 13. The biocompatible material of claim 1 further comprising abiological material.
 14. The biocompatible material of claim 13, whereinthe biological material is selected from the group consisting oferythropoietin, stem cell factor (SCF) vascular endothelial growthfactor (VEGF), transforming growth factor (TGF), fibroblast growthfactor (FGF), epidermal growth factor (EGF), cartilage growth factor(CGF), nerve growth factor (NGF), keratinocyte growth factor (KGF),skeletal growth factor (SGF), osteoblast-derived growth factor (BDGF),hepatocyte growth factor (HGF), insulin-like growth factor (IGF),cytokine growth factor (CGF), stem cell factor (SCF), platelet-derivedgrowth factor (PDGF), endothelial cell growth supplement (EGGS), colonystimulating factor (CSF), growth differentiation factor (GDF), integrinmodulating factor (IMF), calmodulin (CaM), thymidinc kinase (TK), tumornecrosis factor (TNF), growth hormone (GH), bone morphogenic proteins(BMP), matrix metalloproteinase (MMP). tissue inhibitor matrixmetalloproteinase (TIMP), interferon, interleukins, cytokines, integrin,collagen, elastin, fibrillins. fibronectin, laminin, glycosaminoglycans,hemonectin, thrombospondin, heparan sulfate, dermantan, chondrotinsulfate (CS), hyaluronic acid (HA), vitronectin, proteoglycans,transferrin, cytotactin, tenascin, and lymphokines.
 15. Thebiocompatible material of claim 1, wherein the biocompatible material issuitable for implantation into a patient.
 16. A method for treating adefective, diseased, damaged or ischemic tissue or organ in a subjectcomprising implanting the biocompatible material of claim 1 into thesubject.
 17. A method for treating a defective, diseased, damaged orisehemic tissue or organ in a subject comprising injecting thebiocompatible material of claim 1 into the subject.
 18. A method forreconstructing a tissue in a subject comprising implanting thebiocompatible material of claim 1 into the subject.
 19. A method forreconstructing a tissue in a subject comprising injecting thebiocompatible material of claim 1 into the subject.